Flora 274 (2021) 151734 Contents lists available at ScienceDirect Flora journal homepage: www.elsevier.com/locate/¯ora Pollinators of Hoya pottsii: Are the strongest the most effective? Sven Landrein a,1,*, Zi-Yu Zhou a,1, Shi-Jie Song a,b a Xishuangbanna Tropical Botanical Garden, Horticulture department, Menglun, Xishuangbanna, Yunnan, 666303 China b University of the Chinese Academy of Sciences, Beijing 100049 China ARTICLE INFO ABSTRACT Keywords: Hoya floral characters are highly elaborate and associated with a complex and specialised pollination mecha­ Pollination nism. The pollination of two Hoya species has been studied previously, but little is known about the specific Arolium nature and interactions between flowermorphology, pollinators, and their environment. Here we investigate the Erebinae pollination of Hoya pottsii, where pollinaria are transferred onto several insects’ legs and arolia including moths Guide rail in the Erebidae family, ants, and a praying mantis. Hypopyra vespertilio (Erebidae, Erebinae) was the most Hoya carnosa Hoya pottsii effective at both carrying and depositing the pollinaria, Colobopsis leonardii (Formicidae) was shown to suc­ Hypopyra vespertilio cessfully insert only one pollinium whereas Hymenopus coronatus (Hymenopodidae) could only attach the pol­ linaria between its two euplantulae. Several Hoya species were used to compare the effectiveness of pollinaria removal and insertion, pollinator size which was correlated to strength, floral scent, and morphology of the guide rail. The floral scent was dominated by Linalool, Methyl benzoate and Benzaldehyde which are known to attract moth, other species displayed similar scents but also showed many different compounds. The effectiveness of a medium-sized moth in pollinating H. pottsii could be explained by the morphology of the guide rail which comprises a landing platform for the arolium. In Hoya carnosa the guide rail lacks a landing platform which could explain why stronger and larger moths were more effective in this species. The importance of the interaction between insect arolia and guide rails in the pollination of Hoya is illustrated and we suggest that their morphology corresponds with pollinator strength and how smoothly and precisely the pollinia can be inserted. 1. Introduction corona, pollinaria and guide rails, directing pollinators into a precise position within the corolla, and facilitating pollinaria removal and Hoya, with around 300 species is the largest genus in Asclepiadoi­ deposition (Endress 2016). Hoya corona lobes are folded and fused deae tribe Marsdenieae, in the family Apocynaceae. Distributed in SE basally with the anther skirt, forming a sac-like structure where nectar Asia and Australia, with some species in India and Sri Lanka, most secretion is located (Forster and Liddle, 1992; Kunze and Wanntorp, species are epiphytic, succulent vines developing extra-axillary flower 2008). The primary nectary is located inside a slit on the anther skirt; its clusters at the end of a thick persistent peduncle (Kleijn and Van Don­ base forms a tube where the nectar accumulates and its apical part be­ kelaar, 2001; Wanntorp et al., 2014). Apocynaceae are mostly pollinated comes cartilaginous on the sides and forms the guide rail. In most Hoya by insects (Hymenoptera, Diptera, Lepidoptera, Coleoptera, Hemiptera species the primary nectary in the slit has lost its nectar secretion and Neuroptera), with few records of bird pollination (Ollerton and function and is replaced by a secondary nectary formed by the corona Liede, 1997; Ollerton et al., 2009, 2017; Pauw, 1998). The majority of lobes (Wanntorp and kunze, 2009). The pollinaria are formed from the the taxa that have been studied are pollinated by a single functional thecae of adjacent stamens which become fused together, attached by pollinator group (Ollerton et al., 2019). the retinaculum and caudicle (translator arm) to the corpusculum Extreme synorganization of floralparts such as the androecium and (Fig. 1H). The corpusculum is a cartilaginous hollow structure with a gynoecium is characteristic of Apocynaceae (Endress 2016). These fu­ channel in the centre that acts like a clip, attaching to the pollinator sions may have led to the development of other features such as the (Wanntorp, 2007). The pollinia have a thick margin on the inner side * Corresponding author. E-mail addresses: [email protected], [email protected] (S. Landrein). 1 These authors contributed equally. https://doi.org/10.1016/j.flora.2020.151734 Received 5 February 2020; Received in revised form 15 November 2020; Accepted 16 November 2020 Available online 20 November 2020 0367-2530/© 2020 Elsevier GmbH. All rights reserved. S. Landrein et al. Flora 274 (2021) 151734 known as the pellucid margin, this margin gets trapped in the guide rail In this study, we investigated the pollinators of H. pottsii by capturing and pollen tubes will grow externally through the margin to reach the flower visitors during the evening and night. Pollinators were checked stigmatic surface. The guide rail morphology is of great importance in for pollinaria attachment as well as their location on the body and how the pollination process but has received little attention in previous many pollinia were missing, which is indicative of pollination effec­ studies; it is here described in more detail (Fig. 1A). tiveness. Pollinator strength was estimated by using wing length as a The abovementioned floral characters, often associated with polli­ proxy and this was correlated with their respective effectiveness. Guide nator specialisation, have been considered critical to speciation and rails were measured and observed by SEM and compared with Hoya evolutionary radiation (Grant 1949; Stebbins 1970; Crepet 1983). carnosa. Further experiments with living pollinators were performed, However, synorganization evolution in some Apocynaceae taxa has also hand pollination with severed moth legs and tools were explored to resulted in generalised plant-pollinator interactions (Waser et al., 1996). understand the finedetails of such elaborate processes. Floral scent was Although morphological features of Hoya flowers are well under­ extracted and compared with that of three other Hoya species including stood, their function have only been studied in two species, Hoya aus­ H. carnosa, H. incrassata and H. heuschkeliana, to find out if some com­ tralis (Forster, 1992) and H. carnosa (Mochizuki et al., 2017). The two pounds were specific to certain Hoya species. studied species have different pollinators and the studies suggest that Hoya pollination is species specific. Erebus esphesperis (Eribideae), a 2. Materials and methods nocturnal settling moth, was confirmedas the predominant pollinator in H. carnosa and capable of attaching the pollinaria on the aroliar pad. 2.1. Pollinator observations Ocybadistes walkeri, a diurnal skipper butterfly, was shown to be the main pollinator of H. australis, although ForsterFoster (1992) did not The research was conducted within the Xishuangbanna Tropical conduct experiments after dusk. Butterflies were capable of attaching Botanical Garden in Southern Yunnan, China. Hoya pottsii is sponta­ the pollinaria on the legs and proboscis (although no exact region of the neous and epiphytic, growing on several cultivated and subspontaneous leg was mentioned). trees in the garden. The species was selected because many fruits were Hoya species produce white flowers,strong nocturnal fragrance, and produced every year, indicating an efficientpollination system (Fig. 2D). abundant nectar, which is strongly suggestive of moth pollination Foraging insects were observed during day and night but after several (Faegri and van der Pijl, 1979; Altenburger and Matile, 1988; Matile, days’ observations, we inferred an optimal pollination time to be be­ 2006). Floral scents have been well studied in some asclepiad genera tween 2000 h and 2300 h, when pollinators were most abundant and such as Pachycarpus (Shuttleworth and Johnson, 2012), Xysmalobium seen landing on the flowers (Figs. 2A,B,F,G). The species flowered be­ (Shuttleworth and Johnson, 2009), Orbea (Shuttleworth et al., 2017) tween the 13th and 30th of April in both 2018 and 2019 (17 days each and Ceropegia (Heiduk et al., 2016). Floral odour compounds emitted by year) and was observed for 102 h. Insect visitors were caught with a net flowers may provide an insight into the ecology and evolution of polli­ and killed by crushing their abdomen or placing them in alcohol. The nation systems (Dobson, 2006), but despite the large diversity of Hoya, moths were identified using the volumes of Moths of Thailand (Pinra­ studies on Hoya pollination and floral scent are scarce (Jürgens et al., tana, 1990) and iNaturalist (iNaturalist.org, 2019). Although almost all 2010). In flowers of Hoya carnosa the scent is only produced at night the larger moths and moths carrying pollinaria could be identified to (Altenburger and Matile, 1988), although this may differ in other Hoya species, many smaller moths could not be identified accurately (Sup­ species. plementary data 1 and 2). Fig. 1. Floral morphology of Hoya pottsii and pollinaria attachment on different pollinators. (A) Scanning Electronic Microscopy (SEM) detail of the guide rail with the narrowing part shown with # and the primary guide rail shown with *. (B) Corolla section after manual insertion of a pollinium followed by pollen tube germination through the pellucid margin. See arrow for pollen tubes. (C) Hypopyra vespertilio